Synthesis and toxicological study of chitosan-pirul (Schinus molle L.) essential oil nanoparticles on Aspergillus flavus.

In recent years, the study of essential oils as antifungal alternatives and their encapsulation to increase their properties for greater effects has been tested. In this work, nanoparticles of chitosan-Schinus molle L. essential oil (CS-PEO-Np) with a size of 260 ± 31.1 nm were obtained by ionic gelation and evaluated in some growth phases of Aspergillus flavus, a toxigenic fungus. At a concentration of 250 µg/mL of CS-PEO-Np, the A. flavus mycelial growth was inhibited at 97.1% with respect to control, at 96 h of incubation; the germination and viability of spores were inhibited at 74.8 and 40%, respectively, after exposure to 500 µg/mL of these nanomaterials, at 12 h of incubation. The fluorescence images of stained spores with DAPI showed the affectations caused by nanoparticles in the cell membrane, vacuoles and vacuolar content, cell wall, and nucleic acids. For both nanoparticles, CS-Np and CS-PEO-Np, no mutagenic effect was observed in Salmonella Typhimurium; also, the phytotoxic assay showed low-to-moderate toxicity toward seeds, which was dependent on the nanoparticle's concentration. The acute toxicity of CS-PEO-Np to A. salina nauplii was considered low in comparison to CS-Np (control), which indicates that the incorporation of Schinus molle essential oil into nanoparticles of chitosan is a strategy to reduce the toxicity commonly associated with nanostructured materials. The nanoparticulated systems of CS-PEO-Np represent an effective and non-toxic alternative for the control of toxigenic fungi such as A. flavus by delaying the initial growth stage.

Relationship between the Antifungal Activity of Chitosan-Capsaicin Nanoparticles and the Oxidative Stress Response on Aspergillus parasiticus.

The fungus Aspergillus parasiticus is a contaminant in agricultural crops and its eradication involves the indiscriminate use of harmful synthetic pesticides. In the search for antifungal agents of natural origin, chitosan (Q) and capsaicin (C) are coupled in the form of nanoparticles (Np), which can possess a direct application under specific conditions. Due to their small size, Np can cross through the cell wall, taking the cells into a pro-oxidant environment known as "oxidative stress", which presents when the reactive oxygen species (ROS) surpass the number of antioxidants in the cell. In the present investigation, nanoparticles of chitosan (Np Q) and nanoparticles of chitosan-capsaicin (Np QC) with an average diameter of 44.8 ± 20.6 nm and 111.1 ± 14.1 nm, respectively, were synthesized, and there was a zeta potential of + 25.6 ± 0.7 mV and + 26.8 ± 6.1 mV, respectively. The effect of the concentration of Np Q (A, B, C, and D), of Np QC (A, B, C, and D), and capsaicin in a solution (control) was evaluated on the viability of the spores, the accumulation of intracellular ROS, and the morphometric changes of A. parasiticus. Acute toxicity of the Np was determined utilizing bioassays with Artemia salina, and acute phytotoxicity was evaluated in lettuce seeds (Lactuca sativa). According to ROS results, capsaicin (control) did not induce oxidative stress in the cell; otherwise, it was observed to have an elevated (p < 0.05) accumulation of ROS when the concentration of Np Q increased. For both, Np Q and Np QC, an inverse physiological pattern relating spore viability and ROS accumulation in the fungus was found; the viability of spores decreased as the ROS accumulation increased. The spore viability of A. parasiticus diminished upon increasing the concentration of chitosan (0.3−0.4 mg/mL) in the Np, while the intracellular accumulation of ROS increased proportionally to the concentration of the nanomaterials in the treatments of Np Q and Np QC. On the other hand, Np QC presented a lower (p < 0.05) toxicological effect in comparison with Np Q, which indicates that the incorporation of bioactive compounds, such as capsaicin, into nanoparticles of chitosan is a strategy that permits the reduction of the toxicity associated with nanostructured materials.

Phytotoxicity, cytotoxicity, and in vivo antifungal efficacy of chitosan nanobiocomposites on prokaryotic and eukaryotic cells.

Chitosan (CS) nanosystems have potential applications for the control of microorganisms in the medical, environmental, and agrifood fields. In vivo and in vitro assays of CS nanosystems have experienced increased activity due to improved physicochemical properties, biological activity, and reactivity. Hence, it is important to determine whether their application involves toxicological risks. The aim of this study was to evaluate the mutagenic, cytotoxic, phytotoxic, and in vivo antifungal activity of chitosan-pyrrole-2-carboxylic acid nanobiocomposites (CS-PCA). The CS-PCA nanoparticles were synthesized by means of the nanoprecipitation technique with a size and ζ-potential of 502 ± 72 nm and + 54.7 ± 15.0 mV, respectively. According to the Ames test, no evidence of mutagenic activity was observed in Salmonella typhimurium strains. The cytotoxic assay showed that the incorporation of PCA into the CS matrix increased the toxic effect on ARPE-19 cells. However, fluorescence microscopy of ARPE-19 cells did not reveal morphostructural changes allusive to cell injury. CS-PCA exhibited strong phytotoxicity on lettuce seeds and the complete inhibition of seed development. The antifungal assay demonstrated that the CS-PCA delayed Aspergillus niger infection in tomato fruit until day 3; however, its use for the pre-treatment of seeds might exert adverse effects on plant development.

Persistence of the antifungal capacity of a fraction of Jacquinia macrocarpa plant against Fusarium verticillioides after continuous exposure.

This study aimed to determine the ability of Fusarium verticillioides in developing mechanisms to counteract the antifungal effect of a fraction from Jacquinia macrocarpa plant extract (JmAF), as well as the morphological and physiological changes that occur during its exposure. The fungus was exposed to JmAF during consecutive periods. A culture sample was taken weekly to determine radial growth, spore germination and size, and fungal ß-1,3-glucanase activity. The results showed that, in the beginning, the radial growth decreased by 85.8%, and spore germination was delayed. As the exposure continued, the fungus showed a recovery, to some extent, in its original characteristics. However, the radial growth of the fungus continued to be inhibited (42.9%) throughout the experiment (7 weeks). The ß-1,3-glucanase activity also was inhibited by 36.4% during the first week of exposure to JmAF. However, the activity was recovered after 7 weeks of exposure.

Biosorption of copper by immobilized biomass of Aspergillus australensis. Effect of metal on the viability, cellular components, polyhydroxyalkanoates production, and oxidative stress.

Heavy metals are toxic especially when they are introduced into the environment due to anthropogenic activities such as metallurgy, mining, and tanning. Removing these pollutants has become a worldwide concern since they cannot be degraded into nontoxic forms causing extended effects in the ecosystems. The use of an Aspergillus australensis was evaluated in order to remove Cu2+ from simulated wastewater. The fungus was isolated from river sludges contaminated with heavy metals and was first evaluated for the determination of Cu2+ tolerance levels. Microscopic fluorescence analysis was carried out to determine the effect of Cu2+ presence on the viability, cellular components, polyhydroxyalkanoates production, and oxidative stress of the fungus, as a response to the stress caused by exposure to metal. In order to achieve copper removal, the A. australensis biomass was produced using batch cultures, and the mycelium was immobilized on a textile media in order to compare the copper-removal efficiency of live or dead biomass. The optimal values of pH and temperature for biomass production were established by using a surface response analysis. Live immobilized biomass was capable of removing Cu2+ from 1.54 ± 0.19 to 2.66 ± 0.26 mg of copper/ g of dry biomass, while values of 1.93 ± 0.03 to 2.36 ± 0.29 mg of copper/g of dry biomass were observed when dead biomass was used. As was expected, copper removal using biomass varied depending on the pH and temperature used.

Synthesis of chitosan biocomposites loaded with pyrrole-2-carboxylic acid and assessment of their antifungal activity against Aspergillus niger.

A wide variety of chitosan (CS) biomaterials have been loaded with different antimicrobial agents to improve the activity of CS against phytopathogenic fungi. Recently, the antimicrobial activity of 1H-pyrrole-2-carboxylic acid (PCA) has been reported as a secondary metabolite of Streptomyces griseus, which was identified as the main bioactive compound in the biological control. However, it is sensitive to light and its activity against filamentous fungi has not yet been reported. The aim of the present research work was to evaluate the biological activity of CS-PCA biocomposites for the control of Aspergillus niger. CS-PCA biocomposites were obtained through nanoprecipitation. In vitro antifungal activity was determined by viability assay, spore germination, morphometric analysis of spores and hyphae, and the analysis of cellular components by fluorescence microscopy. CS-PCA showed an average size and Z potential of 502 ± 72 nm and + 54.7 ± 15 mV, respectively. Micrographs demonstrated well-distributed biocomposites with an apparently spherical shape. A new signal at 1473 cm-1 in the FT-IR spectrum of the CS-PCA biocomposite was observed, confirming the presence of PCA in the composition of the CS-PCA nanosystem. CS-PCA biocomposites reduced the spores' viability by up to 58%. Effects on fungi morphometry, observed as an increase in the spores' average diameter, swelling, distortion, and an increase in the branching of hyphae, were observed. Fluorescence analysis showed oxidative stress and membrane and cell wall damage, mainly at early growth stages. The inhibitory effect against CS-resistant fungi, such as A. niger, opens a door for the control of CS-sensitive fungi.

Activity of chitosan-lysozyme nanoparticles on the growth, membrane integrity, and ß-1,3-glucanase production by Aspergillus parasiticus.

Synthesis of nanocomposites from antimicrobial biopolymers such as chitosan (CS) and lysozyme (LZ) is an important and promising area in bionanotechnology. Chitosan-lysozyme (CS-LZ) nanoparticles (NPs) were prepared by the nanoprecipitation method, using commercial chitosan of 153 kDa. TEM and dynamic light scattering (DLS) analysis were carried out to evaluate the morphology, size, dispersion, and Z potential. Association efficiency of lysozyme was determined using Coomassie blue assay. The antifungal activity of NPs against Aspergillus parasiticus was evaluated through cell viability (XTT), germination and morphometry of spores, and reducing sugars production; the effects on membrane integrity and cell wall were also analyzed. NPs' size were found in the range of 13.4 and 11.8 nm for CS-LZ and CS NPs, respectively, and high Z potential value was observed in both NPs. Also, high association of lysozyme was presented in the CS matrix. With respect to the biological responses, CS-LZ NPs reduced the viability of A. parasiticus and a strong inhibitory effect on the germination of spores (100% of inhibition) was observed at 24 h in in vitro assays. CS-LZ and CS NPs affected the membrane integrity and the cell wall of spores of fungi with respect to control, which is consistent with the low amount of reducing sugars detected. CS-LZ NPs prepared by nanoprecipitation promise to be a viable and safe alternative for use in biological systems, with a possible low or null impact to humans and biota. However, the potential benefits and the environmental and health implications of NPs need to be globally discussed due to its possible negative effects.

Controlled release matrices and micro/nanoparticles of chitosan with antimicrobial potential: development of new strategies for microbial control in agriculture.

The control of micro-organisms responsible for pre- and postharvest diseases of agricultural products, mainly viruses and fungi, is a problem that remains unresolved, together with the environmental impact of the excessive use of chemicals to tackle this problem. Current efforts are focused on the search for efficient alternatives for microbial control that will not result in damage to the environment or an imbalance in the existing biota. One alternative is the use of natural antimicrobial compounds such as chitosan, a linear cationic biopolymer, which is biodegradable, biocompatible and non-toxic, has filmogenic properties and is capable of forming matrices for the transport of active substances. The study of chitosan has attracted great interest owing to its ability to form complexes or matrices for the controlled release of active compounds such as micro- and nanoparticles, which, together with the biological properties of chitosan, has allowed a major breakthrough in the pharmaceutical and biomedical industries. Another important field of study is the development of chitosan-based matrices for the controlled release of active compounds in areas such as agriculture and food for the control of viruses, bacteria and fungi, which is one of the least exploited areas and holds much promise for future research.

Evaluation of pathological effects in broilers during fumonisins and clays exposure.

This study was conducted to evaluate the possible protector effect of bentonite and zeolite in Bovans chicks fed a diet containing 59 mg kg(-1) of fumonisin B1 (FB1) during 3 weeks. A total of 200 one-day-old male chicks were treated varying the amount of bentonite and zeolite. Chick weight was registered weekly. At the end of the experiment, all the chicks were killed, and the livers were analyzed for gross examination and histopathological changes. Plasmatic activity of alanine amino transferase and aspartate amino transferase (AST) were also determined. Sphinganine and the sphinganine-to-sphingosine ratio in serum were evaluated. Both, bentonite and zeolite showed a protector effect against FB1 adsorption in the digestive tract of chicks. Chicks fed with FB1-contaminated feed, amended either with zeolite or bentonite, were heavier, and no macroscopic lesions were observed in the livers. AST activity might be considered as an indicator for FB1 exposition because AST levels were affected when only FB1 was present in the basal diet. These results indicate that both, zeolite and bentonite can be added into feed to diminish the effects of FB1.

Role of fumonisin B1 on the immune system, histopathology, and muscle proteins of white shrimp (Litopenaeus vannamei).

White shrimps, Litopenaeus vannamei, were tested in two indoor trials to determine the effect of fumonisin B1 on (i) immune response, (ii) histopathology, and, (iii) muscle proteins. Trial 1: (0, 0.5, 0.75 and 1.0µg/g of FB1 levels, 18-day duration; shrimp 5-6g) to evaluate the FB1 effect on the immune system and histopathology response. Trial 2: (0.0, 0.5, 0.75 and 1.0µg/g of FB1 levels, 16-day duration; shrimp 5-6g) to detect FB1 effect on muscle proteins. Prophenoloxidase activity was affected by all FB1 concentrations tested. Both, total haemocyte count and phenoloxidase activity decreased by the 18th day in shrimp exposed to FB1. Marked histological changes in the hepatopancreas of shrimp fed on diet containing FB1, at the all FB1 levels tested, as well as a necrotic tissue were observed. Changes in both, electrophoretic patterns and thermodynamic properties of myosin extracted from shrimp exposed to FB1 were also observed.